1. Field of the Invention
This invention r elates to the field of tissue engineering, and more particularly to a method for chemically acellularizing a biological tissue sample, such as a peripheral nerve.
2. Background Art
Peripheral nerve injuries are exceedingly common, occurring clinically after injury or surgical resection. The resulting nerve gaps can produce significant disability and thus require surgical repair. Primary end-to-end nerve coaptation is the procedure of choice for peripheral nerve reconstruction, but in many circumstances the gap between the proximal and distal stump is too large to allow for a tension-free repair. Under these circumstances, surgeons must employ alternate repair techniques, such as nerve grafting.
Peripheral nerve autografts are the g old standard for nerve grafting procedures and provide the optimal degree of reinnervation when primary end-to-end neurorrhaphy is not an option. However, the functional deficits at the donor site following nerve graft harvest as well as the limited amounts of donor nerve tissue available restrict the use of autografts in many clinical situations.
To avoid the problems inherent in autografting, surgeons have investigated different methods of repair such as termino-lateral neurorrhaphy (TLN) and autogenous vein and plastic chamber conduits. TLN entails suturing the distal stump of a severed nerve to the side of an intact adjacent nerve which is not always available . Autogenous vein grafts and plastic chambers are a plentiful supply of nerve regeneration conduits, yet neither provides the trophic support (nerve growth factor, etc.) necessary for axonal regeneration across nerve gaps greater than 2 cm, leading to poor functional outcomes.
Peripheral nerve allografts provide another alternative for nerve repair. Although allografts overcome the problems associated with the previously mentioned repair methods, nerve allograft rejection becomes a major obstacle. Until long term tolerance to nerve allografts can be induced, this technique requires long term systemic immunosuppression and as a result has limited clinical applications.
Acellular nerve grafts, produced by a variety of techniques, have emerged as a possible alternative to overcome the immnunogenicity of allografts. Previous attempts to create acellular nerve tissue have involved several methods, namely irradiation, fixation, heat treatment, or freezing, prior to grafting in an effort to kill or remove the cellular elements of the nerve allograft and reduce immunogenicity. Peripheral nerve grafts that have been acellularized by such methods have been shown to support axonal regeneration across short distances and to elicit a reduced immune response compared with standard peripheral nerve allografts. However, these acellularization methods cause disruption of the cellular elements of the nerve and may concurrently disrupt the endoneurial tubes, thus reducing the potential for axonal elongation through the nerve graft. In addition, cellular debris remaining after the mechanical disruption process may also elicit an immune response which can adversely affect Schwann cell migration and axonal elongation.
Therefore, it is an object according to the present invention to provide a method for chemically acellularizing a biological tissue sample which does not rely on mechanical cell disruption.
It is a further object according to the present invention to provide a method of acellularization which removes the cellular elements from peripheral nerve tissue while leaving the endoneurial architecture intact.
It is another object according to the present invention to provide an acellularization method which produces an acellularized peripheral nerve construct that will support axonal regeneration across nerve gaps.
It is still another object according to the present invention to provide an acellularization method which produces an acellularized peripheral nerve construct that is nonimmunogenic.
Accordingly, a method is provided for chemically acellularizing a biological tissue sample, such as a peripheral nerve. The method includes disrupting the cell membranes of the biological tissue sample, and then denaturing intracellular proteins within the cells of the tissue sample and removing the denatured proteins from the cells while preserving the extracellular matrix to produce an acellularized tissue construct without using mechanical agents.
In a preferred embodiment, the tissue sample is harvested from a suitable donor, and then submersed in a balanced salt solution, such as Dulbecco""s phosphate buffered saline. The disrupting of cell membranes then includes submersing the biological tissue sample in a solution including glycerol, whereas denaturing and removing intracellular proteins includes submersing the biological tissue in at least one detergent solution. The one or more detergent solutions can comprise ionic detergent solutions and nonionic detergent solutions. Most preferably, the tissue sample is submersed in a succession of ionic and nonionic solutions, where the ionic detergent solutions can include sodium deoxycholate or sodium dodecyl sulfate, and the nonionic detergent solutions can include TRITON(copyright) X-100. In addition, the biological tissue sample is preferably rinsed with distilled water between each solution change. The resulting acellularized tissue construct can then be stored in a physiologic saline solution, and later implanted in a suitable recipient.
The above objects and other objects, features, and advantages of the present invention are readily apparent from the following detailed description of the best mode for carrying out the invention when taken in connection with the accompanying drawings.